Nuclear reprogramming of human skin fibroblasts into induced Pluripotent Stem Cells (iPSCs) has revolutionised the field of regenerative medicine (Takahashi K et al Cell 126, 663-676 (2006); Takahashi K., et al. Cell 131, 861-872 (2007)). However, several obstacles prevent the effective translation of iPSC technology for clinical and industrial applications. These include (i) the low kinetics and efficiency with which fibroblasts can be transformed into iPSCs, hindering their high-throughput generation, (ii) the lack of readily accessible cellular substrates, with non-compromised genomes, that can be isolated from patients (Seki T., et al. (2010) Cell Stem Cell 2; 7(1):11-4; Loh Y. H. et al. (2010) Cell Stem Cell. 2; 7(1):15-9; Staerk J., et al., (2010) Cell Stem Cell. 2; 7(1):20-4) (iii) the relative genomic instability of the iPSCs generated and their genomic differences with their somatic progenitors (Hussein, S. M., et al. Nature. 471(7336):58-62 (2011); Martins-Taylor, K. et al (2011). Nat. Biotechnol. 29, 488-91; Yusa et al (2011) Nature 478, 391-4) and (iv) the inability to easily derive feeder-free iPSCs whilst maintaining their pluripotent potential, which is highly desirable for chemically defined differentiation assays, disease modelling, drug testing and development of therapeutics. In addition, it is desirable that the cellular substrate be clonally derived or capable of clonal expansion prior to reprogramming. This allows the generation of a clonal reference genome, which is essential for subsequent high resolution genetic testing such as array Comparative Genome Hybridisation (aCGH) and genome sequencing comparisons
Several improvements in the efficiency of iPSC generation have been reported since their inception, including the use of progenitor/stem cells as cellular substrates and the inclusion of chemical enhancers in reprogramming protocols, although not all have been demonstrated to work in human samples (Feng B., et al. Cell Stem Cell. 4(4):301-12 (2009)). While compounds such as methylation and deacetylation inhibitors, modified mRNA, siRNAs and vitamin C have been shown to increase the reprogramming efficiency of skin fibroblasts to varying degrees, and may prove useful in translational applications, it would be desirable to manipulate the cellular substrate for reprogramming as little as possible. Moreover, skin cells may carry mutations due to their higher exposure to external environmental conditions (Seki et al 2010; Loh et al 2010; Staerk et al 2010). In addition, for some patients, such as those on anticoagulants or those with conditions that affect wound healing (Shore E. M., et al. Nat. Genet. 38 (5): 525-7 (2006)), skin biopsies are neither practical nor desirable.
Haematopoietic stem cells derived from the bone marrow compartment have been shown to reprogram with higher efficiency than skin fibroblasts in the absence of chemical enhancers, presumably due to a more permissive epigenetic state (Okabe M., et al. Blood. 27; 114(9):1764-7 (2009); 5. Eminli S., et al. Nat. Genet. 41 (9):968-76. (2009)). However obtaining these cells routinely from patients is not trivial, requiring invasive bone marrow aspiration or blood mobilization.
The reprogramming of T-cells and myeloid cells from peripheral blood has also been demonstrated (Seki et al 2010; Loh et al 2010; Staerk et al 2010). These investigators have shown it is possible to circumvent the need for invasive procedures to generate patient specific iPSCs, as blood sampling is routine for almost all patients. However the use of these cells presents at least two major problems: (i) circulating T-cells will possess permanent rearrangements of their genomes following T-cell receptor gene recombination events, thus limiting the potential uses of the iPSC derived from them and (ii) the generation of iPSCs from blood obtained from patients with blood cell related diseases could prove difficult. For example, myeloid derived iPSCs from patients with leukaemia carrying the Philadelphia chromosome rearrangement would not be useful for developing personalised cellular therapies.
Recently, the epigenomic and genomic stability of iPSCs has been called into question (Hussein, S. M., et al. Nature. 471(7336):58-62 (2011); Gore, A., et al. Nature. 471(7336):63-7 (2011); Lister, R., et al. Nature. 471(7336):68-73 (2011)). A variety of iPSCs generated from either fibroblasts or adipose-derived stem cells, via either viral integration or non-integrative methods including episomal or mRNA based expression of the reprogramming factors, were compared at the methylome, chromosome, and exome levels to the parent lines used to generate them, along with hESCs. In summary, all the iPSC lines regardless of the parental cell type or the method used showed independent differences to their parental line, with each other and to hESCs, which were in a relative ground state of pluripotency. This is a major concern that will need to be addressed if iPSCs are to be used in cellular therapies, although with sufficient controls (e.g. repeatability across lines) this need not preclude them from development as models of specific diseases and in drug/toxicology screens, which is an especially important consideration for the pharmaceutical industry.